First detection of Waddlia chondrophila in Africa using SYBR Green real-time PCR on veterinary samples.

Waddlia chondrophila is a strict intracellular microorganism belonging to the order Chlamydiales that has been isolated twice from aborted bovine fetuses, once in USA and once in Germany. This bacterium is now considered as an abortigenic agent in cattle. However, no information is available regarding the presence of this bacterium in Africa. Given the low sensitivity of cell culture to recover such an obligate intracellular bacterium, molecular-based diagnostic approaches are warranted. This report describes the development of a quantitative SYBR Green real-time PCR assay targeting the recA gene of W. chondrophila. Analytical sensitivity was 10 copies of control plasmid DNA per reaction. No cross-amplification was observed when testing pathogens that can cause abortion in cattle. The PCR exhibited a good intra-run and inter-run reproducibility. This real-time PCR was then applied to 150 vaginal swabs taken from Tunisian cows that have aborted. Twelve samples revealed to be Waddlia positive, suggesting a possible role of this bacterium in this setting. This new real-time PCR assay represents a diagnostic tool that may be used to further study the prevalence of Waddlia infection.

Capturing epidermal stemness for regenerative medicine.

The skin is privileged because several skin-derived stem cells (epithelial stem cells from epidermis and its appendages, mesenchymal stem cells from dermis and subcutis, melanocyte stem cells) can be efficiently captured for therapeutic use. Main indications remain the permanent coverage of extensive third degree burns and healing of chronic cutaneous wounds, but recent advances in gene therapy technology open the door to the treatment of disabling inherited skin diseases with genetically corrected keratinocyte stem cells. Therapeutic skin stem cells that were initially cultured in research or hospital laboratories must be produced according strict regulatory guidelines, which ensure patients and medical teams that the medicinal cell products are safe, of constant quality and manufactured according to state-of-the art technology. Nonetheless, it does not warrant clinical efficacy and permanent engraftment of autologous stem cells remains variable. There are many challenges ahead to improve efficacy among which to keep telomere-dependent senescence and telomere-independent senescence (clonal conversion) to a minimum in cell culture and to understand the cellular and molecular mechanisms implicated in engraftment. Finally, medicinal stem cells are expansive to produce and reimbursement of costs by health insurances is a major concern in many countries.

Cellular and viral factors implicated in persistent infection in canine distemper virus

Summary SLAM (signalling lymphocyte activation molecule, CD150) serves as a cellular receptor for different morbiliviruses, including measles virus and canine distemper virus. Laboratory cell lines that do not express dog SLAM are therefore quite refractory to infection by wildtype CDV. SLAM expression is not only required for CDV virion attachment, but also for the establishment of cytolytic infection characterized by syncytia formation. In order to determine if SLAM has a direct influence on CDV replication, we compared wild-type and mutated SLAM variants for their capacity to influence viral polymerase activity and syncytia formation. Deletion of immunoreceptor tyrosine-based signalling motif (ITSM) in the cytoplasmic tail of SLAM did not seem to influence viral replication, viral polymerase activity or cell-to cell fusion. Instead, it was the level of cell surface expression of SLAM, which was important. Additional experiments corroborated the importance of SLAM for efficient cell-to cell fusion: Both SLAM, as well as viral fusion (F) and attachment (H) glycoproteins, were found to be required for efficient cell-to-cell fusion, which, in turn, enhanced the activity of the viral polymerase and, viral replication. Wild-type A75/17 canine distemper virus (CDV) strain is known to induce a persistent infection in the central nervous system and in dog footpad keratinocytes in vivo. Recently, it has been shown that the A75/17 virus could also infect canine footpad keratinocytes (CFKs) in vitro. CFK infection with A75/17 was initially inefficient and produced very little virus progeny, however, after only three passages the adapted virus produced more progeny and induced limited syncytia formation. Sequence comparison between the A75/17 and the CFKadapted A75/17-K virus revealed three amino acid differences, one in the phosphoprotein (P), one in the matrix protein (M) and one in the H protein. In order to identify viral determinants of A75/17-K adaptation, recombinant viruses containing one, two or three nucleotides substitutions were analyzed. The amino acid substitution in the M protein was without effect on viral particle formation. In contrast, the amino acid substitution in the cytoplasmic tail of H protein was clearly important for syncytia formation. Concerning the mutation in the P protein, it led to an increase in viral replication. However, we cannot rule out that the observed effect is due to the amino acid substitutions in the overlapping accessory proteins C and V, also affected by the P mutation. The adaptation of wild-type CDV strains to cell culture almost always involves modifications of M protein. In order to understand the influence of these modifications, we tested recombinant A75/17 viruses bearing different M proteins. Preliminary results demonstrated that the M protein from the Vero-adapted strain reduced syncytia formation. Future studies will focus on the M mRNA and protein stability, its expression level, localisation and its effect on viral particles formation and on the phenotype of infection. Résumé La protéine SLAM (signalling lymphocyte activation molecule ou CD150) est utilisée comme récepteur cellulaire par les morbillivirus parmi lesquels on trouve le virus de la rougeole (VR) ainsi que le virus de la maladie de Carré (CDV). Les lignées cellulaires qui n'expriment pas la protéine SLAM du chien à leur surface sont réfractaires à l'infection par les souches sauvages de CDV. Le récepteur SLAM n'est pas seulement requis pour l'attachement du virion à la surface de la cellule, mais il participe également de façon active à l'établissement d'une infection cytolytique à travers la formation de syncytia. Afin de déterminer si la protéine SLAM exerce une influence directe sur la réplication virale du virus de la maladie de Carré, nous avons généré différentes protéines tronquées de SLAM et comparé leurs capacités à influencer l'activité de la polymérase ainsi que la formation de syncytia. Nos résultas ont montré que la réplication virale, l'activité de la polymérase ainsi que la fusion cellulaire ne semblent pas être influencées par les délétions dans les régions cytoplasmiques du récepteur SLAM. Cependant, ces délétions agissent sur l'expression de la protéine SLAM à la surface des cellules. Les expériences additionnelles ont permis de souligner l'importance de la protéine SLAM dans le phénomène de fusion entre cellules. En effet, la protéine SLAM ainsi que les deux glycoprotéines virales F et H sont requises pour la formation de syncytia, laquelle induit une augmentation de l'activité de la polymérase ainsi que de la réplication virale. La souche virulente A75/17 du virus, de la Maladie de Carré est connue pour induire une infection persistante au niveau du système nerveux central ainsi que dans les kératinocytes de pattes chez le chien. Des études récentes ont montré que des cultures primaires de kératinocytes de chien pouvaient aussi êtres infectées par la souche A75/17 de CDV. En effet, le virus induit une infection persistante en produisant très peu de progéniture. Cependant, trois passages du virus sauvage A75/17 dans ces cultures aboutissent à la sélection d'un virus produisant plus de progéniture et favorisant la formation limitée de syncytia. La comparaison des séquences génomique entre la souche A75/17 et la souche adaptée A75/17-K montre une différence de trois nucléotides. La première mutation, située dans le gène P, modifie la phosphoprotéine (P) ainsi que les protéines V et C. La deuxième se situe dans le gène de la protéine matricielle (M) et la dernière dans celui de la protéine d'attachement (H). Afin de déterminer les facteurs viraux impliqués lors de l'adaptation virale dans la culture primaire de kératinocytes, des virus recombinants contenant une, deux ou trois de ces mutations ont été analysés. La substitution d'un acide aminé dans la protéine M reste sans effet sur la production de particules virales. En revanche, la substitution d'un acide aminé dans la queue cytoplasmique de la protéine H s'avère clairement importante pour la formation de syncytia. Quant à la mutation dans le gène P, elle permet une augmentation de la réplication virale. Cependant, nous ne pouvons pas écarter l'hypothèse que l'augmentation de la réplication virale soit due aux substitutions d'un acide aminé dans les protéines accessoires V et C qui sont, elles aussi, affectées par la mutation dans le gène P. L'adaptation des souches sauvages de CDV aux cultures de cellules induit presque toujours des modifications de la protéine matricielle M. Afin de comprendre l'influence de ces modifications, nous avons testé 'des virus A75/17 recombinants contenant différentes protéines M. Les résultats préliminaires ont démontré que la protéine M de la souche adaptée aux cellules Vero réduisait la formation de syncytia. Les études futures seront axées sur la stabilité de l'ARN messager, celle de la protéine M, de son niveau d'expression, de sa localisation cellulaire et de son effet sur la formation de particules virale ainsi que sur le phénotype de l'infection.

Wound-healing gene family expression differences between fetal and foreskin cells used for bioengineered skin substitutes.

For tissue engineering, several cell types and tissues have been proposed as starting material. Allogenic skin products available for therapeutic usage are mostly developed with cell culture and with foreskin tissue of young individuals. Fetal skin cells offer a valuable solution for effective and safe tissue engineering for wounds due to their rapid growth and simple cell culture. By selecting families of genes that have been reported to be implicated in wound repair and particularly for scarless fetal wound healing including transforming growth factor-beta (TGF-beta) superfamily, extracellular matrix, and nerve/angiogenesis growth factors, we have analyzed differences in their expression between fetal skin and foreskin cells, and the same passages. Of the five TGF-beta superfamily genes analyzed by real-time reverse transcription-polymerase chain reaction, three were found to be significantly different with sixfold up-regulated for TGF-beta2, and 3.8-fold for BMP-6 in fetal cells, whereas GDF-10 was 11.8-fold down-regulated. For nerve growth factors, midkine was 36-fold down-regulated in fetal cells, and pleiotrophin was 4.76-fold up-regulated. We propose that fetal cells present technical and therapeutic advantages compared to foreskin cells for effective cell-based therapy for wound management, and overall differences in gene expression could contribute to the degree of efficiency seen in clinical use with these cells.

Calmodulin kinase IV: expression and function during rat brain development.

The expression of calmodulin kinase IV (CaMKIV) can be induced by the thyroid hormone T3 in a time- and concentration-dependent manner at a very early stage of brain differentiation using a fetal rat telencephalon primary cell culture system which can grow and differentiate under chemically defined conditions (Krebs et al. (1996) J. Biol. Chem. 271, 11055-11058). After the induction of CaMKIV by T3 we examined the influence of prolonged absence of T3 from the culture medium on the expression of CaMKIV. We could demonstrate that after the T3-dependent induction of CaMKIV, omission of the hormone, even for 8 days, from the medium did not downregulate the expression of CaMKIV indicating that different regulatory mechanisms became important for the expression of the enzyme. We further showed that CaMKIV could be involved in the Ca(2+) -dependent expression of the immediate early gene c-fos, probably via phosphorylation of the transcription factor CREB. Convergence of signal transduction pathways on this transcription factor by using different protein kinases may explain the importance of CREB for the regulation of different cellular processes.

Recovery of a persistent Canine distemper virus expressing the enhanced green fluorescent protein from cloned cDNA.

Wild-type A75/17-Canine distemper virus (CDV) is a highly virulent strain, which induces a persistent infection in the central nervous system (CNS) with demyelinating disease. Wild-type A75/17-CDV, which is unable to replicate in cell lines to detectable levels, was adapted to grow in Vero cells and was designated A75/17-V. Sequence comparison between the two genomes revealed seven nucleotide differences located in the phosphoprotein (P), the matrix (M) and the large (L) genes. The P gene is polycistronic and encodes two auxiliary proteins, V and C, besides the P protein. The mutations resulted in amino acid changes in the P and V, but not in the C protein, as well as in the M and L proteins. Here, a rescue system was developed for the A75/17-V strain, which was shown to be attenuated in vivo, but retains a persistent infection phenotype in Vero cells. In order to track the recombinant virus, an additional transcription unit coding for the enhanced green fluorescent protein (eGFP) was inserted at the 3' proximal position in the A75/17-V cDNA clone. Reverse genetics technology will allow us to characterize the genetic determinants of A75/17-V CDV persistent infection in cell culture.

Ultraweak photon emission in assessing bone growth factor efficiency using fibroblastic differentiation.

Photons participate in many atomic and molecular interactions and changes. Recent biophysical research has shown the induction of ultraweak photons in biological tissue. It is now established that plants, animal and human cells emit a very weak radiation which can be readily detected with an appropriate photomultiplier system. Although the emission is extremely low in mammalian cells, it can be efficiently induced by ultraviolet light. In our studies, we used the differentiation system of human skin fibroblasts from a patient with Xeroderma Pigmentosum of complementation group A in order to test the growth stimulation efficiency of various bone growth factors at concentrations as low as 5 ng/ml of cell culture medium. In additional experiments, the cells were irradiated with a moderate fluence of ultraviolet A. The different batches of growth factors showed various proliferation of skin fibroblasts in culture which could be correlated with the ultraweak photon emission. The growth factors reduced the acceleration of the fibroblast differentiation induced by mitomycin C by a factor of 10-30%. In view that fibroblasts play an essential role in skin aging and wound healing, the fibroblast differentiation system is a very useful tool in order to elucidate the efficacy of growth factors.

Vitamin D Receptor and Jak-STAT Signaling Crosstalk Results in Calcitriol-Mediated Increase of Hepatocellular Response to IFN-α.

Recent clinical research suggests a role for vitamin D in the response to IFN-α-based therapy of chronic hepatitis C. Therefore, we aimed to explore the underlying mechanisms in vitro. Huh-7.5 cells harboring subgenomic hepatitis C virus (HCV) replicons or infected with cell culture-derived HCV were exposed to bioactive 1,25-dihydroxyvitamin D3 (calcitriol) with or without IFN-α. In these experiments, calcitriol alone had no effect on the HCV life cycle. However, calcitriol enhanced the inhibitory effect of IFN-α on HCV replication. This effect was based on a calcitriol-mediated increase of IFN-α-induced gene expression. Further mechanistic studies revealed a constitutive inhibitory interaction between the inactive vitamin D receptor (VDR) and Stat1, which was released upon stimulation with calcitriol and IFN-α. As a consequence, IFN-α-induced binding of phosphorylated Stat1 to its DNA target sequences was enhanced by calcitriol. Importantly, and in line with these observations, silencing of the VDR resulted in an enhanced hepatocellular response to IFN-α. Our findings identify the VDR as a novel suppressor of IFN-α-induced signaling through the Jak-STAT pathway.

Differential stress reaction of human colon cells to oleic-acid-stabilized and unstabilized ultrasmall iron oxide nanoparticles.

Therapeutic engineered nanoparticles (NPs), including ultrasmall superparamagnetic iron oxide (USPIO) NPs, may accumulate in the lower digestive tract following ingestion or injection. In order to evaluate the reaction of human colon cells to USPIO NPs, the effects of non-stabilized USPIO NPs (NS-USPIO NPs), oleic-acid-stabilized USPIO NPs (OA-USPIO NPs), and free oleic acid (OA) were compared in human HT29 and CaCo2 colon epithelial cancer cells. First the biophysical characteristics of NS-USPIO NPs and OA-USPIO NPs in water, in cell culture medium supplemented with fetal calf serum, and in cell culture medium preconditioned by HT29 and CaCo₂ cells were determined. Then, stress responses of the cells were evaluated following exposure to NS-USPIO NPs, OA-USPIO NPs, and free OA. No modification of the cytoskeletal actin network was observed. Cell response to stress, including markers of apoptosis and DNA repair, oxidative stress and degradative/autophagic stress, induction of heat shock protein, or lipid metabolism was determined in cells exposed to the two NPs. Induction of an autophagic response was observed in the two cell lines for both NPs but not free OA, while the other stress responses were cell- and NP-specific. The formation of lipid vacuoles/droplets was demonstrated in HT29 and CaCo₂ cells exposed to OA-USPIO NPs but not to NS-USPIO NPs, and to a much lower level in cells exposed to equimolar concentrations of free OA. Therefore, the induction of lipid vacuoles in colon cells exposed to OA utilized as a stabilizer for USPIO NPs is higly amplified compared to free OA, and is not observed in the absence of this lipid in NS-USPIO NPs.

Cultured 3T3L1 adipocytes dispose of excess medium glucose as lactate under abundant oxygen availability

White adipose tissue (WAT) produces lactate in significant amount from circulating glucose, especially in obesity;Under normoxia, 3T3L1 cells secrete large quantities of lactate to the medium, again at the expense of glucose and proportionally to its levels. Most of the glucose was converted to lactate with only part of it being used to synthesize fat. Cultured adipocytes were largely anaerobic, but this was not a Warburg-like process. It is speculated that the massive production of lactate, is a process of defense of the adipocyte, used to dispose of excess glucose. This way, the adipocyte exports glucose carbon (and reduces the problem of excess substrate availability) to the liver, but the process may be also a mechanism of short-term control of hyperglycemia. The in vivo data obtained from adipose tissue of male rats agree with this interpretation.

Placental growth factor-1 and epithelial haemato-retinal barrier breakdown: potential implication in the pathogenesis of diabetic retinopathy.

AIMS/HYPOTHESIS: Disruption of the retinal pigment epithelial (RPE) barrier contributes to sub-retinal fluid and retinal oedema as observed in diabetic retinopathy. High placental growth factor (PLGF) vitreous levels have been found in diabetic patients. This work aimed to elucidate the influence of PLGF-1 on a human RPE cell line (ARPE-19) barrier in vitro and on normal rat eyes in vivo. METHODS: ARPE-19 permeability was measured using transepithelial resistance and inulin flux under stimulation of PLGF-1, vascular endothelial growth factor (VEGF)-E and VEGF 165. Using RT-PCR, we evaluated the effect of hypoxic conditions or insulin on transepithelial resistance and on PLGF-1 and VEGF receptors. The involvement of mitogen-activated protein kinase (MEK, also known as MAPK)/extracellular signal-regulated kinase (ERK, also known as EPHB2) signalling pathways under PLGF-1 stimulation was evaluated by western blot analysis and specific inhibitors. The effect of PLGF-1 on the external haemato-retinal barrier was evaluated after intravitreous injection of PLGF-1 in the rat eye; evaluation was by semi-thin analysis and zonula occludens-1 immunolocalisation on flat-mounted RPE. RESULTS: In vitro, PLGF-1 induced a reversible decrease of transepithelial resistance and enhanced tritiated inulin flux. These effects were specifically abolished by an antisense oligonucleotide directed at VEGF receptor 1. Exposure of ARPE-19 cells to hypoxic conditions or to insulin induced an upregulation of PLGF-1 expression along with increased transcellular permeability. The PLGF-1-induced RPE cell permeability involved the MEK signalling pathway. Injection of PLGF-1 in the rat eye vitreous induced an opening of the RPE tight junctions with subsequent sub-retinal fluid accumulation, retinal oedema and cytoplasm translocation of junction proteins. CONCLUSIONS/INTERPRETATION: Our results indicate that PLGF-1 may be a potential regulation target for the control of diabetic retinal and macular oedema.

Nanoparticles for gene delivery to retinal pigment epithelial cells.

PURPOSE: To evaluate the safety and potential use of poly(lactic) acid (PLA) and poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) as vectors for gene transfer to RPE cells. METHODS: Experiments were conducted with primary bovine RPE cells and with the ARPE-19 human RPE cell line. Rhodamine loaded NPs were used to study factors influencing the internalization process by the various RPE cells: concentrations of NPs, duration of contact time, stage of cell culture and ambient temperature. The extent of NPs internalization was evaluated by fluorescence and phase microscopy. Potential NP toxicity was measured by the trypan blue exclusion dye test and the MTT method. Green fluorescent protein (GFP) plasmid or red nuclear fluorescent protein (RNFP) plasmid were sequestered in NPs. The ability ot these "loaded" NPs to generate gene transfection and protein expression in RPE cells was assessed both in vivo and in vitro by fluorescence and confocal microscopy. RESULTS: The extent of NP internalization in cultured cells increases with their concentration reaching a plateau at 1 mg/ml and a contact time of up to 6 h. Temperature and culture stage did not influence the in vitro internalization process. No toxic effects on RPE cells could be detected when these were incubated with up to 4 mg/ml of NPs. In human and bovine RPE cells incubated with GFP loaded NPs, cytoplasmic green fluorescence was observed in 14+/-1.65% of the cultured cells. Incubation with RNFP loaded NPs yielded a nuclear red fluorescence in 18.9+/-1.6% of the cells. These percentage levels of expression initially detected after 48 h of incubation remained unchanged during the following 8 additional days in culture. No significant differences in the extent of cytoplasm or nuclear fluorescence expression were observed between bovine or human RPE cultured cells. In vivo, a preferential RNFP expression within the RPE cell layer was detected after intra vitreous injection of RNFP plasmid loaded NPs. CONCLUSIONS: The ability of PLGA NPs to sequester plasmids, their nontoxic characteristics, and rapid internalization enables gene transfer and expression in RPE cells. These findings may be of potential use when designing future gene therapy strategies for ocular diseases of the posterior segment.

Searching for Novel Cellular Targets of the Hepatitis C Virus NS3-4A Protease

Background: The hepatitis C virus (HCV) NS3-4A protease isnot only an essential component of the viral replication complexand a prime target for antiviral intervention but also a key playerin the persistence and pathogenesis of HCV. It cleaves andthereby inactivates two crucial adaptor proteins in viral RNAsensing and innate immunity (MAVS and TRIF) as well as aphosphatase involved in growth factor signaling (TC-PTP). Theaim of this ongoing study is to identify novel cellular targets ofthe NS3-4A protease.Methods: Cell lines inducibly expressing the NS3-4A proteasewere established using a tetracycline-regulated geneexpression system. Cells were analyzed in basal as well asinterferon-α-stimulated states. Two-dimensional difference gelelectrophoresis (2D-DIGE) and stable isotopic labeling usingamino acids in cell culture (SILAC) proteomics analysescoupled with mass spectrometry were employed to search forcellular substrates of NS3-4A.Results: A number of candidate cellular targets have beenidentified by these proteomics approaches. These are currentlybeing validated by different experimental techniques. In parallel,we are in the process of further defining the determinants forsubstrate specificity of the NS3-4A protease.Conclusions: The identification of novel cellular targets of theHCV NS3-4A protase should yield new insights into thepathogenesis of hepatitis C and may reveal novel targets forantiviral intervention.

Mycobacterium tuberculosis subverts innate immunity to evade specific effectors.

The macrophage is the niche of the intracellular pathogen Mycobacterium tuberculosis. Induction of macrophage apoptosis by CD4(+) or CD8(+) T cells is accompanied by reduced bacterial counts, potentially defining a host defense mechanism. We have already established that M. tuberculosis-infected primary human macrophages have a reduced susceptibility to Fas ligand (FasL)-induced apoptosis. To study the mechanisms by which M. tuberculosis prevents apoptotic signaling, we have generated a cell culture system based on PMA- and IFN-gamma-differentiated THP-1 cells recapitulating the properties of primary macrophages. In these cells, nucleotide-binding oligomerization domain 2 or TLR2 agonists and mycobacterial infection protected macrophages from apoptosis and resulted in NF-kappaB nuclear translocation associated with up-regulation of the antiapoptotic cellular FLIP. Transduction of a receptor-interacting protein-2 dominant-negative construct showed that nucleotide-binding oligomerization domain 2 is not involved in protection in the mycobacterial infection system. In contrast, both a dominant-negative construct of the MyD88 adaptor and an NF-kappaB inhibitor abrogated the protection against FasL-mediated apoptosis, showing the implication of TLR2-mediated activation of NF-kappaB in apoptosis protection in infected macrophages. The apoptosis resistance of infected macrophages might be considered as an immune escape mechanism, whereby M. tuberculosis subverts innate immunity signaling to protect its host cell against FasL(+)-specific cytotoxic lymphocytes.

Cultured 3T3L1 adipocytes dispose of excess medium glucose as lactate under abundant oxygen availability

White adipose tissue (WAT) produces lactate in significant amount from circulating glucose, especially in obesity;Under normoxia, 3T3L1 cells secrete large quantities of lactate to the medium, again at the expense of glucose and proportionally to its levels. Most of the glucose was converted to lactate with only part of it being used to synthesize fat. Cultured adipocytes were largely anaerobic, but this was not a Warburg-like process. It is speculated that the massive production of lactate, is a process of defense of the adipocyte, used to dispose of excess glucose. This way, the adipocyte exports glucose carbon (and reduces the problem of excess substrate availability) to the liver, but the process may be also a mechanism of short-term control of hyperglycemia. The in vivo data obtained from adipose tissue of male rats agree with this interpretation.